Field
The present disclosure is directed to the creation of variant or mutagenized herpes viruses and host cells containing, including but not limited to SVV, HSV-1, HSV-2, VZV, CMV, EBV, HHV6, HH7 and KSHV/HHV8, which variants or mutagenized viruses are rendered conditionally replication defective by the incorporation or fusion of one or more destabilization domains onto one or more genes which are essential for viral replication. In an exemplary embodiment the present invention is directed to a recombinant varicella virus (VV) strain and methods for the construction thereof, wherein the virus absent modification is prone to becoming latent or dormant in the ganglia and reactivating to cause zoster or shingles, and wherein the varicella virus, preferably varicella zoster virus (VZV) or simian varicella virus (SVV), is modified to render the virus conditionally replication deficient, i.e., the virus only replicates under defined conditions.
In an exemplary embodiment the present invention is directed to a recombinant herpes simplex virus 1 or 2 strain and methods for the construction thereof, wherein the virus is similarly modified to render the virus conditionally replication deficient, i.e., the virus only replicates under defined conditions, such as by the incorporation or fusion of one or more destabilization domains onto one or more genes essential for virus replication, e.g., the DNA polymerase gene.
The invention further specifically relates to the use of the resultant conditional replication defective VZV strains in vaccine compositions in order to immunize individuals against shingles and zoster, especially in order to prevent shingles in individuals who would otherwise be susceptible to reactivation of VZV and the onset of shingles e.g., based on advanced age, cancer, or immunodeficiency such as HIV-AIDS or another disorder resulting in impaired T cell function.
In another exemplary embodiment the present invention is directed to recombinant herpes simplex 1 or 2 (HSV-1, HSV-2) virus strains, cells and vaccines containing, and methods for the construction thereof, wherein the virus is rendered conditionally replication defective by the incorporation or fusion of a destabilization domain onto one or several genes essential for HSV-2 replication, e.g., the DNA polymerase and/or ICP4 genes. In a related aspect it is an object to provide vaccine compositions containing and the use thereof in order to immunize individuals against these herpes viruses.
Description of Related Art
Varicella Zoster Virus: Varicella zoster virus (VZV) causes chickenpox (varicella) in children. After varicella, VZV becomes latent in ganglia along the entire neuraxis (Mahalingam et al., 1990) and spontaneously reactivates decades later resulting in zoster (shingles), characterized by pain and rash restricted to 1-3 dermatomes. Each year, 600,000 to 1 million Americans are affected by zoster (NIH Shingles Prevention Study: www.niaid.nih.gov).
In the aging population, although VZV-specific humoral immunity is intact (Gershon and Steinberg, Am. J. Med. Sci. 1981 July-August;282(1):12-7), a decline in cell-mediated immunity to VZV (Miller, Neurology. 1980 June;30(6):582-7) correlates with the incidence of zoster (Arvin A M. Varicella-zoster virus. In: Knipe D M, Howley P, editors. Fields' virology. 4th edn. Philadelphia: Lippincott-Williams & Wilkins; 2001a. pp. 2731-2768; Oxman et al., 2005). The development of zoster may be viewed in the context of a continuum in immunodeficiencies, ranging from a natural decline in VZV-specific T cell immunity with age, to more serious immune deficiencies seen in cancer patients, organ transplant recipients, and ultimately in AIDS patients. Operationally defined as pain persisting for more than 4 to 6 weeks after zoster, PHN is the most common complication of zoster. PHN develops in >40% zoster patients over age 60 (200,000-300,000 Americans/year). Zoster patients also develop stroke from uni- or muli-focal vasculopathy, as well as myelitis and zoster paresis, retinitis and even pain without rash.
VZV is the first human herpesvirus for which a live-attenuated vaccine (OKA) is routinely administered to children in the USA (Arvin and Gershon, 1996). A more potent VZV vaccine has also been shown to reduce the incidence of zoster. An extensive study in 38,000 humans >60 years showed that zostavax vaccine reduced the number of zoster cases by 51% and the occurrence of PHN by 66% (Oxman et al., 2005). The FDA approved the vaccine for healthy adults >60 years. By the year 2030, it is estimated that 22% of the US citizens (65 million people) will be >65 years and by 2050, at least 21 million will be >85 years (Quan et al., 2007). Thus, in an ideal situation, even if every human >60 years were vaccinated, there would still be at least 500,000 zoster patients and almost half of whom will experience PHN. VZV myelitis and vasculopathy and possibly PHN are caused by persistent VZV infection (Gilden et al., Neuropathol. Appl. Neurobiol. 2011 August; 37(5):441-463).
OKA vaccine virus reactivates asymptomatically in vaccinated individuals (Krause and Klinman, 2000). Alternative strategies to develop new VZV vaccines include heat-inactivated as well as subunit vaccine (Breuer, 2006). Heat inactivated vaccine induces reduced class I-restricted killing of virus-specific lymphocytes (Hayward et al., Virol Immunol. 1996;9(4):241-5). Candidate zoster vaccines based on a recombinant (truncated) form of the VZV glycoprotein E (gE) boosts a pre-existing anti-gE humoral response (Vafai 1995). However, injection of mice with VZV gE peptides does not produce a strong humoral immune response (Breuer 2001). Such a vaccine is currently being tested with specific adjuvants as a target to boost cell-mediated immunity (Dendouga et al., 2012). A more efficient vaccine that induces a strong immune response but is less likely to reactivate is needed. In this context, a conditional varicella mutant (using a gene such as ORF 63 that is required for replication) can be used as a vaccine. Such a vaccine virus will provide a good humoral as well as cell-mediated immune response but would not reactivate to produce zoster.
Attempts to Produce VZV Infection in Animals.
VZV causes disease only in humans. Development of an experimental animal model that recapitulates the pathogenesis of VZV in humans is the goal of several laboratories. Corneal inoculation of mice (Wroblewska et al., 1993), intra-muscular inoculation of guinea pigs (Lowry et al., 1993; Tenser and Hyman 1987; Chen et al., 2003; Sadzot-Delvaux et al., 1990), and food pad inoculation of rats (Debrus et al., 1995; Kennedy et al., 2001) with VZV results in sero-conversion and virus entry into ganglia, confirmed by the detection of VZV DNA and RNA, without disease. VZV inoculation of human thymus and ganglionic implants under the kidney capsule of SCID-hu mice results in virus infection (Moffat et al., J. Virol. 1995 September;69(0):5236-42; Zerboni et al., Virology. 2005 Feb. 5;332(1):337-46). However, VZV reactivation has not been demonstrated in any of these models. Recently inoculation of cynomolgus monkeys with VZV has been shown to result in mild immune response without overt clinical symptoms although ganglia were not analyzed for the presence of latent VZV (Willer et al., J. Virol. 2012 April;86(7):3626-34).
Simian Varicella Virus (SVV) as a Model for Human VZV Disease.
Immunological, virological and pathological features of SVV infection of non-human primates closely resemble those of human VZV infection. Like VZV in humans, primary infection of primates with SVV leads to varicella followed by virus latency and spontaneous reactivation. Various non-human primate subspecies are susceptible to SVV infection (Gray, Rev. Med. Virol. 2004 November-December;14(6):363-81; Mahalingam and Gilden, In: Arvin A, Campadelli-Fiume G, Mocarski E, et al., editors. Human Herpesviruses: Biology, Therapy, and Immunoprophylaxis. Cambridge: Cambridge University Press; 2007; Chapter 58). SVV infection of rhesus macaques (RM) closely parallels VZV infection in humans, providing the best model of human VZV infection (Messaoudi et al., PLoS Pathog. 2009 November;5(11):e1000657). Primary SVV infection in monkeys results in viremia followed by rapid proliferation of both B and T cells (Ouwendijk et al., J. Neurovirol. 2012 April; 18(2):91-99; Ouwendijk et al., PloS Pathog. 2013 May;9(5):e1003368). SVV-specific IgG antibodies are detected at 10 dpi and peak at 17-19 days. Cell-mediated immune responses to SVV develop by day 7, with peak T cell counts seen at approximately 14 days (Messaoudi et al., 2009; Ouwendijk et al., 2012; Ouwendijk et al., 2013). Virus-specific T cell populations are a mixture of interferon-y-producing (effector) CD4+ T cells and CD8+ T cells and appear early during the immune response in correlation with the development of SVV-specific antibodies. CD8+ T cells have increased amounts of granzyme B, indicating cytotoxic potential (Messaoudi et al., 2009). After varicella, SVV becomes latent in ganglionic neurons (Kennedy et al. Virus Genes. 2004 April;28(3):273-6) at all levels of the neuraxis (Mahalingam et al, J. Virol. 2002 September;76(17):8548-8550). The present inventors have demonstrated SVV reactivation after social and environmental stress and after immunosuppression by irradiation, tacrolimus or prednisone (Mahalingam et al., Virology. 2007 Sep. 30;36(2):387-93; Mahalingam et al., J. Neurovirol. 2010 October;16(5):342-54). When SVV infection is disseminated, liver and lung are the most affected organs. Inflammation, hemorrhagic necrosis and eosinophilic intranuclear inclusions are seen in affected skin and viscera. SVV and VZV share immunological cross-reactivity. Sequence analysis of the complete SVV genome revealed 75% DNA homology with VZV (Gray et al., Virology. 2001 May 25;284(1):123-30). The complete SVV genome is available as a bacterial artificial chromosome (BAC) (Gray et al., Arch. Virol. 2011 May;156(5):739-46). Point mutations can be introduced to elucidate the role of specific SVV genes in pathogenesis, latency and reactivation (Mahalingam et al., 2011).
Varicella ORF 63 Protein.
VZV ORF 63, which is present as a duplicate copy as ORF 70 on the VZV genome, is the most abundant transcript in latently infected human ganglia, and the protein is present in cytoplasm of infected neurons (Cohrs and Gilden, J. Virol 2007 March;81(6):2950-6; Mahalingam et al., Proc. Natl. Acad. Sci. U.S.A. 1996 Mar. 5;93(5):2122-4). A recent study of human ganglia latently infected with VZV has shown that the number of neurons expressing VZV ORF 63/70 protein during latency is much less than previously reported (Zerboni et al., J. Virol. 2010 April; 84(7):3421-3430). VZV ORF 63 protein is a component of the VZV tegument and represses both VZV as well as cellular promoters (Bontems et al., J. Biol. Chem. 2002 Jun. 7; 277(23):21050-60; Di Valentin et al., Biol. Chem. 2005 March; 386(3):255-67). VZV ORF 63/70 protein inhibits α-interferon-induced antiviral resonse in non-neuronal cells in culture (Ambagala and Cohen, J. Virol 2007 August; 81(15):7844-7851), alters the ability of human anti-silencing function 1 protein to bind histones (Ambagala et al., J. Virol. 2009 January; 83(1):200-209), and inhibits neuronal apoptosis in cultured human ganglia (Hood et al., J. Virol. 2006 January; 80(2): 1025-1031). VZV ORF 63/70 protein binds to RNA polymerase II and VZV IE 62 and enhances VZV gI protomer activity (Lynch et al., Virology. 2002 Oct. 10;302(1):71-82). SVV ORF 63/70 shares 52% amino acid identity with VZV ORF 63/70 (Gray et al., 2001). Like VZV, SVV ORF 63/70 is also transcribed and translated in latently infected monkey ganglia (Mahalingam et al., 1996; Messaoudi et al., 2009). Recently, the inventors showed that while SVV ORF 63/70 is not essential for the development of cytopathic effect (CPE), productive replication of SVV lacking ORF 63/70 is impaired in Vero cells (Brazeau et al., J. Neurovirol. 2011 June; 17(3):274-280). These results, obtained using BAC constructs for the first time to examine this requirement of SVV ORF 63/70, were consistent with the findings of Cohen et al. 2004 in VZV (Cohen et al., J. Virol. 2004 November; 78(21):11833-11840). Cohen et al. 2004 purported but did not demonstrate that a VZV mutated to delete ORF 63 could afford advantages to current live VZV vaccines.
Therefore, notwithstanding the foregoing, there exists a great need for improved VZV vaccines, especially those which elicit long-lived protective immunity (ideally over an entire lifetime) and which are not subject to the virus sequestering in the ganglia and reactivating in susceptible individuals resulting in the onset of zoster or shingles. Also, because of the general problem of different herpesviruses becoming latent in specific cells of infected individuals, and viral infection reoccurring, e.g., in the case of HSV-1 and HSV-2, the problem of reoccurring oral or genital lesions, there is a great need for safe and effective herpesvirus vaccines, most especially for affording immuno protection against HSV-1 and HSV-2.